WO2013155686A1 - 整合素β亚基在诊断静脉血栓栓塞中的应用 - Google Patents

整合素β亚基在诊断静脉血栓栓塞中的应用 Download PDF

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WO2013155686A1
WO2013155686A1 PCT/CN2012/074284 CN2012074284W WO2013155686A1 WO 2013155686 A1 WO2013155686 A1 WO 2013155686A1 CN 2012074284 W CN2012074284 W CN 2012074284W WO 2013155686 A1 WO2013155686 A1 WO 2013155686A1
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integrin
subunit
substance
vte
acute
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PCT/CN2012/074284
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English (en)
French (fr)
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王乐民
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Wang Lemin
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Priority to JP2015506062A priority Critical patent/JP5989893B2/ja
Priority to PCT/CN2012/074284 priority patent/WO2013155686A1/zh
Priority to EP12874796.1A priority patent/EP2840396B1/en
Priority to US14/395,216 priority patent/US9863958B2/en
Publication of WO2013155686A1 publication Critical patent/WO2013155686A1/zh
Priority to HK15103035.4A priority patent/HK1202626A1/zh

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2842Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2845Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/7055Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/70553Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
    • G01N2333/70557Integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00Labels used in chemical analysis of biological material
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/22Haematology
    • G01N2800/226Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Definitions

  • the present invention relates to the field of venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • the present invention provides a novel method of diagnosing VTE comprising detecting levels of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in a blood sample of a subject.
  • the invention further relates to the use of a substance capable of specifically binding an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit in the preparation of a diagnostic agent for the diagnosis of VTE.
  • the present invention provides a kit for diagnosing VTE, which comprises a substance capable of specifically binding an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit. Background technique
  • Venous thromboembolism is a general term for thrombosis formed in the vein, including pulmonary thromboembolism ( ⁇ ) and deep vein thrombosis (DVT), including acute pulmonary embolism ( ⁇ ), chronic thromboembolic pulmonary hypertension (CTEPH). ).
  • VTE is a frequently-occurring disease, in which PE has become a worldwide health care problem due to its high morbidity, high misdiagnosis rate and high mortality rate (Prevention and treatment of venous thromboembolism. International Consensus Statement (guidelines according to scientific evidence). Int. Angiol. 2006;25(2): 101-161).
  • venous vascular endothelial cell injury or hyperfunction of blood coagulation factors damaged endothelial cells and platelets are activated, clotting factor function is activated, and venous thrombosis is formed.
  • the thrombus formed in the vein is rich in fibrin and red blood cells, and has only a small amount of platelets (Furie, Bruce; Furie, Barbara, Mechanisms of Thrombus Formation, The New England Journal of Medicine 2008; 359 (9): 938-949) .
  • the protein component of the APE thrombus is mainly composed of fibrinogen, which also contains a small amount of serum protein and cytoskeletal protein (Wang L, Gong Z, Jiang J, Xu W, Duan Q, Liu J, et al. Confusion of Wide Thrombolytic Time Window for Acute Pulmonary Embolism: Mass Spectrographic Analysis for Thrombus Proteins. Am. J. Respir. Crit. Care Med. 2011 184: 145-146).
  • This hair It has changed the traditional view that venous thrombosis protein is mainly fibrin, which provides a new way for the diagnosis and treatment of VTE.
  • studies of the manner in which fibrinogen in VTE thrombus binds to leukocytes, platelets, and red blood cells in a thrombus can provide information on the mechanism of formation of venous thrombosis.
  • Integrins are a class of receptor proteins that mediate the attachment of cells and cells to their surrounding tissues, such as other cells or extracellular matrices. It also plays an important role in cell signaling and thereby mediates the exchange of information between cells and the surrounding environment, allowing cells to respond quickly to changes in the environment (eg, clotting blood through platelets) (Hynes R, Integrins : bidirectional, allosteric signaling machines. Cell (2002), 110 (6): 673-87).
  • Integrins are heterodimers that contain two distinct chains, called (X subunits and beta subunits. Currently, 18 alpha subunits and 9 beta subunits are identified in mammalian inteins, By combining these subunits, more than 20 integrins can be constructed (Humphries MJ. Integrin structure. Biochem Soc Trans. 2000; 28(4): 311-39). These 20 integrins constitute the integrin family. The integrin containing the same ⁇ subunit constitutes the integrin ⁇ subfamily.
  • the integrin ⁇ 1-3 subunits encoded by the ITGB1, /7UB2, and ITGB3 genes are ITGB1 (also known as CD29), ITGB2 (again It is called CD18), and ITGB3 (also known as CD61).
  • ITGB1 also known as CD29
  • ITGB2 also known as CD18
  • ITGB3 also known as CD61
  • the integrin ⁇ subfamily containing the ITGB1 subunit is widely distributed, mainly mediating the adhesion between cells and extracellular matrix components, mediating lymphocyte homing and white blood cells. Adhesion to activated endothelial cells; ⁇ subfamily is mainly distributed on the surface of activated lymphocytes and platelet membranes (Billard MJ, Mclntyre BW.
  • the integrin-containing ⁇ 2 subfamily is mainly found on various leukocyte surfaces and is used to mediate cell-cell interactions, which are mainly distributed in neutrophils, In the monocytes (Fu C, Tong C, Wang M, Gao Y, Zhang Y, Lu S, Liang S, Dong C, Long M. Determining beta2-integrin and intercellular adhesion molecule 1 binding kinetics in tumor cell adhesion to leukocytes and J Biol Chem.
  • the invention relates to the use of a substance that specifically binds to integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in the preparation of a diagnostic agent for the diagnosis of venous thromboembolism.
  • the invention relates to a substance that specifically binds to an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit in the preparation of an integrin ⁇ subunit for use in detecting a blood sample of a subject
  • a level of integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit to diagnose a diagnostic agent for venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • the blood sample of the invention is a peripheral blood sample, preferably a peripheral venous blood sample.
  • the invention relates to a substance that specifically binds at least two of an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit in the preparation of a blood sample for use in detecting a subject
  • a substance that specifically binds at least two of an integrin beta ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in the preparation of a blood sample for use in detecting a subject
  • the invention relates to a substance that specifically binds to an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and an integrin ⁇ 3 subunit, respectively, in preparing a corresponding integrin ⁇ subunit for use in detecting a blood sample of a subject , the integrin ⁇ 2 subunit, and the elevated levels of the integrin ⁇ 3 subunit, thereby diagnosing the use of diagnostic agents for venous thromboembolism.
  • the venous thromboembolism of the invention is acute pulmonary embolism (APE), chronic thromboembolic pulmonary hypertension (CTEPH), or deep vein thrombosis (DVT).
  • APE acute pulmonary embolism
  • CTEPH chronic thromboembolic pulmonary hypertension
  • DVT deep vein thrombosis
  • the venous thromboembolism of the invention is acute pulmonary embolism (APE) or acute deep vein thrombosis (DVT).
  • the substance of the invention that specifically binds to a ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit is an antibody, ligand or antagonist.
  • the antibodies, ligands, and/or antagonists of the invention are labeled.
  • the antibodies, ligands, and/or antagonists of the invention are fluorescently labeled.
  • the antibody, ligand, and/or antagonist of the invention is a phycoerythrin ( ⁇ )-labeled antibody.
  • the invention provides a method for detecting venous thromboembolism (VTE) by detecting levels of integrin ⁇ ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in a blood sample of a subject, such as a peripheral venous blood sample. ) kit.
  • the invention provides a method for diagnosing venous thromboembolism by detecting levels of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in a blood sample of a subject, such as a peripheral venous blood sample.
  • a kit (VTE) comprising a substance that specifically binds to an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit, respectively.
  • the kit of the invention comprises a substance that specifically binds to at least two of the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit. In one embodiment, the kit of the present invention comprises a substance that specifically binds to the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and the integrin ⁇ 3 subunit, respectively.
  • the substance specifically binding to the ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit included in the kit of the present invention is an antibody, a ligand, and/or an antagonist. .
  • the antibodies, ligands, and/or antagonists of the invention are labeled.
  • the antibodies, ligands, and/or antagonists of the invention are fluorescently labeled.
  • the antibody, ligand, and/or antagonist of the invention is a phycoerythrin ( ⁇ )-labeled antibody.
  • FIG. 1 Shotgun protein sample pre-separation results.
  • Figure 2 Results of the thrombin protein sample enzymatic peptide and fragment sequence information.
  • Figure 3 Serial protein profiling and information analysis of thrombus samples from patients with acute sputum, Gene Oncology analysis.
  • Figure 4 Serial protein profiling and information analysis of thrombus samples in patients with acute PE. The results of interaction network analysis showed that ⁇ 2, ⁇ 3 and ⁇ subunit integrins are the core proteins of red thrombus.
  • Figure 5 Serial protein profile and information analysis of thrombus samples in patients with acute lower extremity VTE, Gene
  • Figure 6 Serial protein profiling and information analysis of thrombus samples in patients with acute lower extremity VTE, interaction network analysis results, indicating that ⁇ 3 subunit integrin and ITGA2B (ie integrin ⁇ chain lib) are the core proteins of red thrombus.
  • Figure 7 Distribution of integrin ⁇ 1, ⁇ 2, and ⁇ 3 subunits in healthy controls.
  • Figure 7a shows that the integrin ⁇ subunit is normally distributed
  • Figure 7b shows that the integrin ⁇ 2 subunit is non-normal.
  • Distribution Figure 7c shows that the integrin ⁇ 3 subunit is normally distributed.
  • Figure 8 Expression of integrin ⁇ subunit in a healthy population control group.
  • Figure 8a shows The mean expression and baseline interval of integrin ⁇ subunit in the control group, with a mean of 8.18 ⁇ 2.69 (%) and a baseline interval of 2.8-13.56 (%).
  • Figure 8b shows the distribution of integrin ⁇ subunit expression levels in the control group.
  • Figure 9 Expression of integrin ⁇ 2 subunit in a healthy control group.
  • Figure 9a shows the mean and baseline interval of integrin ⁇ 2 subunit expression in the control group, with a median expression of 88.6 ⁇ %) and a baseline interval of 71.1-95.8 (%).
  • Figure % shows the distribution of integrin ⁇ 2 subunit expression levels in the control group.
  • Figure 10 Expression of integrin ⁇ 3 subunit in a healthy control group.
  • Figure 10a shows the mean and baseline interval of integrin ⁇ 3 subunit expression in the control group, with a mean of 9.65 ⁇ 2.53 (%) and a baseline interval of 4.59-14.71 (%).
  • Figure 10b shows integrin ⁇ 3 in the control group. The distribution of base expression levels. Detailed description of the invention
  • the present invention provides a novel method of diagnosing or treating venous thromboembolism, which comprises detecting or modulating integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or in a blood sample of a subject.
  • the level of the integrin ⁇ 3 subunit is also relates to a substance capable of specifically binding, detecting, or modulating an integrin beta subunit, and the use of the substance in the preparation of a kit for diagnosing VTE.
  • thrombus refers to a blood clot formed in a blood vessel that interferes with or blocks blood flow.
  • blood platelets and certain proteins aggregate to form blood clots to repair damaged sites.
  • the blood clot falls off, it may become a blood clot.
  • venous thromboembolism refers to a general term for thrombus formed in veins, including pulmonary thromboembolism ( ⁇ ) and deep vein thrombosis (DVT), including acute pulmonary embolism (APE), chronic thromboembolic pulmonary artery. High pressure (CTEPH). Deep vein thrombosis includes acute and chronic deep vein thrombosis.
  • integrin refers to a class of cell surface receptor proteins. Such proteins are heterodimers that contain two distinct chains, referred to as the alpha subunit and the beta subunit, respectively. An integrin containing the same beta subunit constitutes the integrin beta subfamily.
  • the ⁇ subfamily containing the integrin ⁇ subunit also known as CD29
  • the ⁇ 2 subfamily containing the integrin ⁇ 2 subunit also known as CD18
  • ⁇ 3 containing the integrin ⁇ 3 subunit also known as CD61
  • the alpha subunit and the beta subunit encompass variants formed by differential splicing, such as four variants of ITGB1.
  • differential splicing refers to the process of producing different mRNA splice isoforms by different splicing means (i.e., combining exons by different splice sites) in the same mRNA precursor. Protein products obtained by alternative splicing are isotypes of each other, which may exhibit different functional and structural properties, or may result in different phenotypes in the same cell due to different expression levels.
  • subject refers to a mammalian individual, wherein human is preferred.
  • the invention provides a novel method of diagnosing venous thromboembolism.
  • the method of the invention comprises detecting the level of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit in a blood sample of a subject, and detecting the level of the test with a healthy subject A comparison is made between the corresponding levels measured in the control blood samples, wherein the elevated levels are indicative of the diagnosis of VTE.
  • blood sample refers to a blood tissue sample taken from a subject's circulatory system, preferably a blood sample taken from a peripheral vein.
  • the blood sample of the present invention may contain blood cells and extracellular fluid (i.e., blood plasma).
  • the "level increase" in the present invention can be determined, for example, by determining a range of normal levels in a healthy population by using a certain number of healthy people as a control group, and then if the level of the object to be measured is higher than the normal The level of the level is considered to be the level of the object to be tested.
  • the normal range of levels in healthy people this can be determined by statistical methods. For example, the Kolmogorov-Smirnov test is used for the normality test, and the P>0.05 is a normal distribution.
  • the normal range is determined by the average measured in the healthy population combined with its standard deviation ⁇ S).
  • the normal level range is represented by the median combined with P2.5-P97.5 in statistics.
  • level rise may mean, for example, a measured level and blood of a healthy subject.
  • the control level in the liquid sample is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95%, 100% or even higher.
  • Healthy subject refers to a group of subjects whose physical examination is normal.
  • Control level refers to the level of integrin-related protein detected from a healthy subject population, and then a statistical average is taken and used as a control level in combination with the standard deviation.
  • the population of healthy subjects used for sampling as a control may for example be 50 objects, 100 objects, 150 objects, 200 objects, 250 objects, 300 objects, 500 objects or even more.
  • the method of the method of the present invention comprises extracting a blood sample, such as a peripheral venous blood sample, from the subject to be tested.
  • the amount of each integrin beta subunit in the blood sample is then quantitatively detected using a substance that specifically binds to various integrin beta subunits, such as an antibody, wherein the method for quantitative detection can be, for example, flow cytometry, ELISA And other conventional techniques.
  • Peripheral blood samples of several (e.g., 200 or more) healthy subjects were extracted, and the amount of each integrin ⁇ subunit was separately detected by the same method as above, and then statistical average values were taken and combined with standard deviation as a control level.
  • the amount of each integrin beta subunit measured in the subject is compared to the corresponding control level, wherein elevation of various integrin beta subunit levels is a characterization for the diagnosis of VTE.
  • the method of the invention comprises detecting the level of at least two of the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit in the blood sample of the subject. In one embodiment, the method of the invention comprises detecting the level of at least two of the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit in the blood sample of the subject. In one embodiment, the method of the invention comprises detecting the levels of integrin ⁇ subunit, integrin ⁇ 2 subunit, and integrin ⁇ 3 subunit in a blood sample of a subject.
  • the venous thromboembolism described in the present invention may be acute pulmonary embolism ( ⁇ ), chronic thromboembolic pulmonary hypertension (CTEPH), or deep vein thrombosis (DVT), preferably acute pulmonary embolism or deep vein thrombosis.
  • acute pulmonary embolism
  • CTEPH chronic thromboembolic pulmonary hypertension
  • DVT deep vein thrombosis
  • the methods of the invention detect levels of integrin beta ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit by using flow cytometry.
  • the invention provides a substance for specifically detecting the level of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit.
  • the invention provides a substance that specifically binds to an integrin beta subunit, an integrin beta 2 subunit, and/or an integrin beta 3 subunit, such as an antibody, a ligand, and/or Antagonist.
  • the antibodies, ligands and/or antagonists of the invention are labeled. In one embodiment, the antibodies, ligands and/or antagonists of the invention are fluorescently labeled. In one embodiment, the antibodies, ligands and/or antagonists of the invention are labeled with phycoerythrin ( ⁇ ).
  • the ligand of the integrin ⁇ subunit is, for example, laminin (LM), collagen (COL), fibronectin (FN), vascular cell adhesion molecule-1 (VCAM-1) and the like.
  • Ligands of the integrin ⁇ 2 subunit are, for example, fibrinogen (FB), intercellular adhesion molecule-1, intercellular adhesion molecule-2, intercellular adhesion molecule-2 (ICAM-1, ICAM-2, ICAM). -3), X factor, ic3b, etc.
  • Ligands of the integrin ⁇ 3 subunit include, for example, FB, FN, vitronectin (VN), von Willebrand factor (vWF), thrombin sensitive protein (TSP), and the like.
  • the invention relates to a substance which specifically binds to an integrin ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit in the preparation of an integrin ⁇ subunit for use in detecting a blood sample of a subject, integration Use of the level of the ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit to diagnose a diagnostic agent for venous thromboembolism.
  • the invention relates to the preparation of a substance for specifically diagnosing venous thromboembolism by specifically binding at least two of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit, respectively. Use in the agent.
  • the invention relates to at least two substances which specifically bind at least two of a ⁇ subunit, an integrin ⁇ 2 subunit, and/or an integrin ⁇ 3 subunit, respectively, for preparation for diagnosis of venous thromboembolism Use in the composition.
  • the substance that specifically binds at least two of the ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit, respectively refers to at least two different substances, one of which is specific for the substance sexually binds to one of the ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, and the other substance specifically binds to the ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit. Another one.
  • the invention relates to the use of a substance that specifically binds to a ⁇ subunit, an integrin ⁇ 2 subunit, and an integrin ⁇ 3 subunit, respectively, in the preparation of a composition for the diagnosis of venous thromboembolism.
  • the substance that specifically binds to the ⁇ subunit, the integrin ⁇ 2 subunit, and the integrin ⁇ 3 subunit, respectively refers to at least three different substances, wherein the first substance specifically binds to the ⁇ subunit Or one of the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, the second substance specifically binding to the ⁇ subunit, the integrin ⁇ 2 subunit, or the second of the integrin ⁇ 3 subunit, and the The three substances specifically bind to the ⁇ subunit, the integrin ⁇ 2 subunit, or the third of the integrin ⁇ 3 subunits.
  • the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit can be specifically bound to be used for specific detection of integrin ⁇ subunit, integrin ⁇ 2 in blood samples.
  • Substances at the subunit, and/or integrin ⁇ 3 subunit levels are antibodies, antagonists and/or ligands, preferably labeled antibodies, antagonists and/or ligands.
  • the antibodies, antagonists and/or ligands of the invention are fluorescently labeled, e.g., labeled with phycoerythrin ( ⁇ ).
  • the invention provides a kit for diagnosing venous thromboembolism, comprising a substance for specifically detecting levels of integrin ⁇ ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit Wherein the substance is a substance capable of specifically binding to the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit.
  • the kit of the invention comprises a substance that specifically binds to at least two of the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit.
  • the kit of the invention comprises a substance that specifically binds at least two of the integrin ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, respectively.
  • the kit of the invention comprises a substance that specifically binds to the integrin ⁇ subunit, the integrin ⁇ 2 subunit, and the integrin ⁇ 3 subunit, respectively.
  • the invention also relates to ligands, antibodies, antagonists and/or activity inhibitors of integrin ⁇ subunit, integrin ⁇ 2 subunit, and/or integrin ⁇ 3 subunit.
  • ligand As used in the present invention are terms commonly used in the art, and the meanings thereof are generally understood by those skilled in the art, and can also be referred to commonly used. The definition of textbooks and manuals.
  • a substance that specifically binds at least two of the ⁇ subunit, the integrin ⁇ 2 subunit, and/or the integrin ⁇ 3 subunit, respectively refers to at least two different substances, one of which is specific for the substance. Binding to one of the ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, and the other substance specifically binds to the ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit another.
  • Specific binding to the ⁇ subunit, the integrin ⁇ 2 subunit, and the integrin ⁇ 3 subunit, respectively Quality refers to at least three different substances, the first of which specifically binds to one of the ⁇ subunit, the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, and the second substance specifically binds to ⁇ a second of the integrin ⁇ 2 subunit, or the integrin ⁇ 3 subunit, and the third substance specifically binds to the ⁇ subunit, the integrin ⁇ 2 subunit, or the third of the integrin ⁇ 3 subunit .
  • the invention also relates to a device for performing a diagnosis using the method of the invention.
  • a device for performing a diagnosis using the method of the invention.
  • a device that can perform clinical rapid diagnosis.
  • Example 1 Sampling Acute VTE Thrombosis with a Catheter:
  • the femoral vein puncture method was used to obtain the thrombus length from a pulmonary artery of a 39-year-old male acute PE (APE) patient and a male 50-year-old acute lower extremity femoral vein (ADVT) using a 7F right coronary artery catheter (Metronic, USA).
  • Fresh blood clots of 15 mm and 10 g of red blood clots were used for the tandem protein profiling in the following examples.
  • Example 2 Tandem protein profiling and data information analysis methods:
  • the peptide data were analyzed by bioinformatics to accurately identify the protein corresponding to the peptide fragment, and the protein was classified and analyzed by biological pathway.
  • the GO database contains functional information on the biological processes involved in the genes, the location of the cells in which they are located, and the molecular functions they perform. Differentially expressed proteins were used as input to GO analysis. The differential genes are mapped to the respective nodes of the GO database. Calculate the number of genes per node. 1.3 KEGG Pathway analysis method:
  • KEGG pathway analysis was performed on differentially expressed proteins.
  • GErel gene expression interactions indicate a relationship between transcription factors and target gene products.
  • the interaction between protein and protein can be obtained from MIPS data (htt : ⁇ mips.helmholtz-muenchen.de/proj/ppi/).
  • the co-citation algorithm is used to download the abstract of the PubMed database, break down each digest into sentences, and then analyze the co-existing gene names in each sentence, ie, the co-referenced genes. For each pair of co-introduced gene pairs, the frequency of occurrence of the pair of genes is counted. The higher the frequency of gene pairs, the greater the possibility of interaction between the pair of genes.
  • the total number of PubMed database documents is denoted as N, and the frequencies of the two genes in the gene pair independently appear in the PubMed literature database are recorded as ⁇ n respectively.
  • the actual number of genes occurring simultaneously is fc, based on the hypergeometric distribution, and the probability of more than k co-references under completely random conditions is calculated:
  • Example 2 The APE and ADVT thrombus samples obtained in Example 1 were subjected to mass spectrometry, and the mass spectrometry model was LTQ (Thermo Finnigan USA).
  • a FinniganTM LTQTM Linear Ion Capture MS (Thermo Electron) equipped with an electrospray interface was attached to the LC device for detection of eluted peptides. At the same time, a data-dependent MS/MS spectrum was obtained.
  • Each scan cycle consists of the following: A full Profile mode MS scan followed by 5 Centroid mode MS/MS scans with Dynamic ExclusionTM set as follows: Repeat count 2, repeat time 30 s, exclude time 90 s. Each sample was analyzed in triplicate.
  • the MS/MS spectra were automatically searched for non-redundant International Protein Index (IPI) Human Protein Database (version 3.26, 67687 entries) using Bioworks Browse rev. 3.1 (Thermo Electron, San Jose, CA.).
  • IPI International Protein Index
  • the results of protein identification were extracted from the SEQUEST output file using BuildSummary (Dai J, Shieh CH, Sheng QH, Zhou H, Zeng R: Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps. Anal Chem).
  • the peptide is restricted to the form of the tryptic peptide and allows for up to two lost cracks.
  • the carbamoylation of cysteine is considered a fixed modification, while the oxidation of a methionine residue is considered a variable modification.
  • the allowable mass tolerances are: 2.0 Da for the precursor ion and 0.8 Da for the fragment ion.
  • Protein identification criteria are based on Delta CN (>0.1) and cross-correlation scores (Xcor) One charge ⁇ 1.9, two charges ⁇ 2.2, three charges ⁇ 3.75).
  • Table 1 APE thrombus samples GO analysis results (column diagram in Figure 3)
  • Protein interaction network analysis Simultaneous integration of three different types of interactions to obtain a network of protein interactions, ie, protein interaction, gene regulation, protein modification, etc. in KEGG signaling pathway analysis data; (b) Existing high-throughput experiments, such as yeast-two-hybrid and other confirmed protein-protein interactions; (c) the interactions between genes and proteins mentioned in the literature.
  • the diagnosis of PE and DVT has imaging (CT, MRA, ECT or angiography) evidence and clinical manifestations.
  • the standard can be referred to (Torbicki A, Perrier A, Konstantinides S, et al. Guidelines on the diagnosis and management of acute pulmonary Embolism: the Task Force for the Diagnosis and Management of Acute Pulmonary Embolism of the European Society of Cardiology (ESC) [J]. Eur Heart J, 2008, 29(18): 2276-2315; and Qaseem A, Snow V, Barry P, et al. Current diagnosis of venous thromboembolism in primary care: a clinical practice guideline from the American Academy of Family Physicians and the American College of Physicians [J]. Ann Intern Med, 2007, 146(6): 454-458) .
  • CD61 integrin ⁇ 2 subunit
  • type is mouse anti-human IgG1, K, clone number VI-PL2, concentration is 50 mg/L
  • CD29-PE (pi ), CD18-PE (P2), CD61-PE (P3) fluorescent antibodies are available from BD, USA (Becton, Dickinson and Company, USA);
  • Hemolysin 500 ⁇ l was added to the test tube; the hemolysin used was used to dissolve red blood cells, model OptiLyse C, available from Beckman-Coulter, USA.
  • ⁇ buffer (0.01 M PBS, ⁇ 7.2) was added for flow cytometry.
  • ⁇ buffer (0.01 M PBS, ⁇ 7.2) was added for flow cytometry.
  • the US BECKMAN-COULTER EPICS XL-4 flow cytometer is used;
  • sheath liquid containing active ingredient of sodium sulfate 9.84 g/L, sodium chloride 4.07 g/L, and procaine hydrochloride 0.11 g/L
  • sheath liquid containing active ingredient of sodium sulfate 9.84 g/L, sodium chloride 4.07 g/L, and procaine hydrochloride 0.11 g/L
  • the control group was statistically analyzed using SPSS18 statistical software.
  • the normality test was performed using the Kolmogorov-Smirnov test, and the normal distribution was obtained at P>0.05.
  • the normal distribution data is expressed as ⁇ S, and the comparison between groups of different age groups is analyzed by ANOVA.
  • PO.05 indicates The difference was statistically significant.
  • Non-normal distribution data were expressed as median and P2.5-P97.5.
  • Wilcoxon rank sum test was used for comparison between different sex groups.
  • PO.05 showed statistically significant difference.
  • Kmskal-Wallis H was used for comparison among different age groups. Test, PO.05 indicates that the difference is statistically significant.
  • the normal range is determined by the mean measured in a healthy population combined with its standard deviation ⁇ s).
  • the normal level range is represented by the median combined with P2.5-P97.5 in the statistics.
  • the results are expressed as a percentage of cells (positive cells) showing the corresponding integrin subunit in the total cell count (i.e., the percentage of positive cells in 10,000 cells). See Figure 7-10 and Table 3 below.
  • the integrin ⁇ 1 and ⁇ 3 subunits showed a normal distribution, and the integrin ⁇ 2 subunit showed a non-normal distribution. There was no significant difference in the results between different genders.
  • the ⁇ 1 and ⁇ 3 decreased with age.
  • the baseline interval of integrin ⁇ subunit was 2.8-13.56%, and the integrin ⁇ 2 subunit baseline interval was 71.1-95.8%. Integrin ⁇ 3
  • the subunit baseline interval was 4.59-14.71%.
  • ⁇ and ⁇ 3 increased by 1/24 (4.2%)
  • ⁇ 2 and ⁇ 3 increased by 1/24 (4.2%).
  • the above cytological results indicate that high expression of integrin beta subfamily in VTE patients can be used to diagnose VTE.
  • the results in the above examples indicate that the expression of hematopoietic integrin ⁇ 1, ⁇ 2 and ⁇ 3 subunits in healthy populations is detectable, the expression of ⁇ and ⁇ 3 subunits is normally distributed, and the expression of ⁇ 2 subunits is non-normally distributed. There was no statistical difference between the sexes. The expression of ⁇ and ⁇ 3 subunits decreased with age.

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Abstract

本发明提供了一种诊断静脉血栓栓塞(VTE)的方法,其中包括检测血液样品中的整合素β1亚基、整合素β2亚基、和/或整合素β3亚基的水平。还提供了用于诊断VTE的试剂盒,其中包括能够特异性地结合整合素β1亚基、整合素β2亚基、和/或整合素β3亚基的物质。

Description

整合素 β亚基在诊断静脉血栓栓塞中的应用 技术领域
本发明涉及静脉血栓栓塞 (VTE)领域。 具体而言, 本发明提供了一种新 的诊断 VTE的方法, 其中包括检测对象血液样品中的整合素 βΐ亚基、 整 合素 β2亚基、 和 /或整合素 β3亚基的水平。 本发明还涉及能够特异性地结 合整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的物质在制备用于 诊断 VTE的诊断剂中的用途。此外,本发明还提供了用于诊断 VTE的试剂 盒, 其中包括能够特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整 合素 β3亚基的物质。 背景技术
静脉血栓栓塞是指在静脉中形成的血栓的总称, 其包括肺动脉血栓栓 塞症 (ΡΕ)和深静脉血栓形成 (DVT), 其中 ΡΕ包括急性肺栓塞 (ΑΡΕ)、慢性血 栓栓塞性肺动脉高压 (CTEPH)。 VTE是一种多发病, 其中 PE由于其高发病 率、 高误诊率和高死亡率已经成为世界范围内的卫生保健难题 (Prevention and treatment of venous thromboembolism. International Consensus Statement (guidelines according to scientific evidence). Int. Angiol. 2006;25(2): 101-161)。
根据传统的静脉血栓形成理论 (phlebothrombosis theory),在血流缓慢状 态下, 静脉血管内皮细胞损伤或凝血因子功能亢进, 损伤的内皮细胞和血 小板被活化, 凝血因子功能被激活, 静脉血栓由此形成。 其中静脉内所形 成的血栓富含纤维蛋白以及红细胞, 仅具有少量的血小板 (Furie, Bruce; Furie, Barbara, Mechanisms of Thrombus Formation, The New England Journal of Medicine 2008;359 (9):938-949)。
本发明人最近的研究结果表明, APE 血栓中的蛋白组分主要由纤维蛋 白原组成, 其中还包含少量的血清蛋白和细胞骨架蛋白 (Wang L, Gong Z, Jiang J, Xu W, Duan Q, Liu J, et al. Confusion of Wide Thrombolytic Time Window for Acute Pulmonary Embolism: Mass Spectrographic Analysis for Thrombus Proteins. Am. J. Respir. Crit. Care Med. 2011 184: 145-146)。 这一发 现改变了静脉血栓蛋白主要是纤维蛋白这一传统观点, 为 VTE的诊断和治 疗提供了新的途径。例如,对 VTE血栓中的纤维蛋白原与血栓中的白细胞, 血小板和红细胞之间结合方式的研究, 则可以提供涉及静脉血栓的形成机 制的信息。
整合素是介导细胞与细胞,细胞与其周围组织 (如其它细胞或胞外基质) 之间的附着的一类受体蛋白。 其在细胞信号转导中也发挥着重要作用, 并 由此介导细胞与周围环境的信息交换, 使得细胞可以对环境的变化作出快 速应答 (例如使血液通过血小板而凝结) (Hynes R, Integrins: bidirectional, allosteric signaling machines. Cell (2002), 110 (6): 673—87)。
整合素是异二聚体, 其含有两个不同的链, 分别称为 (X亚基和 β亚基。 目前在哺乳动物的整合素中共鉴别出 18个 α亚基和 9个 β亚基, 通过这些 亚基的分别组合, 可构成 20多种整合素 (Humphries MJ. Integrin structure. Biochem Soc Trans. 2000;28(4):311 -39)。这 20多种整合素构成了整合素家族, 其中含有同一种 β 亚基的整合素构成了整合素 β 亚族。 例如由 ITGB1、 /7UB2、和 ITGB3基因编码的整合素 β1-3亚基分别为 ITGB1(又称作 CD29)、 ITGB2(又称作 CD18)、和 ITGB3(又称作 CD61)。含有 ITGBl亚基的整合素 βΐ亚族分布广泛, 主要介导细胞与细胞外基质成分之间的附着, 介导淋巴 细胞的归巢以及白细胞与激活的内皮细胞间的粘附; βΐ亚族主要分布于激 活的淋巴细胞和血小板膜表面上 (Billard MJ, Mclntyre BW. CD45RA T-cell activation without proliferation by a partial agonist monoclonal antibody to betal integrin.Immunol Cell Biol. 2008 May-Jun;86(4):381-4)。 含有整合素 β2 亚族主要存在于各种白细胞表面, 用于介导细胞间的相互作用, 其主要分 布于中性粒细胞、 单核细胞中 (Fu C, Tong C, Wang M, Gao Y, Zhang Y, Lu S, Liang S, Dong C, Long M.Determining beta2-integrin and intercellular adhesion molecule 1 binding kinetics in tumor cell adhesion to leukocytes and endothelial cells by a gas-driven micropipette assay. J Biol Chem. 2011 Oct 7;286(40):34777-87) o 含有整合素 β3 亚族主要分布于血小板的表面, 其介 导血小板的聚集, 以及参与血栓的形成 (Sachs UJ, Bakchoul T, Eva O, Giptner A, Bein G, Aster RH, Gitter M, Peterson J, Santoso S. A point mutation in EGF-4 domain of β3 integrin is responsible for the formation of the Seca platelet alloantigen and affects receptor function. Thromb Haemost. 2011 ; 107(1》。 发明概述
一方面, 本发明涉及特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3 亚基的物质在制备用于诊断静脉血栓栓塞的诊断剂中的用 途。
在一个实施方案中, 本发明涉及特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的物质在制备用于检测对象血液样品中的整 合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的水平从而诊断静脉血 栓栓塞 (VTE)的诊断剂中的用途。
在一个实施方案中, 本发明的血液样品是外周血样品, 优选外周静脉 血样品。
在一个实施方案中, 本发明涉及特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的物质在制备用于检测对象血 液样品中相应的整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基中至 少两种的水平从而诊断静脉血栓栓塞的诊断剂中的用途。
在一个实施方案中, 本发明涉及分别特异性地结合整合素 βΐ亚基、 整 合素 β2亚基、 和整合素 β3亚基的物质在制备用于检测对象血液样品中相 应的整合素 βΐ亚基、 整合素 β2亚基、 和整合素 β3亚基的升高水平, 从而 诊断静脉血栓栓塞的诊断剂中的用途。
在一个实施方案中, 本发明中所述的静脉血栓栓塞是急性肺栓塞 (APE), 慢性血栓栓塞性肺动脉高压 (CTEPH)、 或深静脉血栓形成 (DVT)。
在一个实施方案中, 本发明中所述的静脉血栓栓塞是急性肺栓塞 (APE) 或急性深静脉血栓形成 (DVT)。
在一个实施方案中, 本发明中特异性地结合 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的物质是抗体、 配体或拮抗剂。在一个实施方案中, 本 发明的抗体、 配体、 和 /或拮抗剂是经标记的。 在一个实施方案中, 本发明 的抗体、 配体、 和 /或拮抗剂是经荧光标记的。 在一个实施方案中, 本发明 的抗体、 配体、 和 /或拮抗剂是经藻红蛋白 (ΡΕ)标记的抗体。
另一方面, 本发明提供了一种检测对象血液样品例如外周静脉血样品 中的整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的水平从而诊断 静脉血栓栓塞 (VTE)的试剂盒。 在一个实施方案中, 本发明提供了一种检测对象血液样品例如外周静 脉血样品中的整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的水平 从而诊断静脉血栓栓塞 (VTE)的试剂盒,其含有分别特异性地结合整合素 βΐ 亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的物质。
在一个实施方案中,本发明的试剂盒包括特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的物质。 在一个实施方 案中, 本发明的试剂盒中包括分别特异性地结合整合素 βΐ亚基、整合素 β2 亚基、 和整合素 β3亚基的物质。
在一个实施方案中,本发明的试剂盒中所包括的特异性地结合 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的物质是抗体、 配体、 和 /或拮抗剂。 在一个实施方案中, 本发明的抗体、 配体、 和 /或拮抗剂是经标记的。 在一 个实施方案中, 本发明的抗体、 配体、 和 /或拮抗剂是经荧光标记的。 在一 个实施方案中, 本发明的抗体、 配体、 和 /或拮抗剂是经藻红蛋白 (ΡΕ)标记 的抗体。 附图说明
图 1 : 血栓蛋白样品鸟枪法预分离结果。
图 2: 血栓蛋白样品酶解肽段、 碎片序列信息结果。
图 3 : 急性 ΡΕ 患者血栓样品串联蛋白质譜和信息分析, Gene Oncology分析结果。
图 4: 急性 PE患者血栓样品串联蛋白质譜和信息分析,相互作用网 络分析结果, 其表明 β2、 β3和 βΐ亚基整合素是红色血栓的核心蛋白。
图 5 : 急性下肢 VTE患者血栓样品串联蛋白质譜和信息分析, Gene
Oncology分析结果。
图 6: 急性下肢 VTE患者血栓样品串联蛋白质譜和信息分析, 相互 作用网络分析结果, 其表明 β3亚基整合素和 ITGA2B (即整合素 α链 lib)是 红色血栓的核心蛋白。
图 7: 整合素 β1、 β2和 β3亚基在健康人群对照组中的分布情况, 其中图 7a表明整合素 βΐ亚基呈正态性分布, 图 7b表明整合素 β2亚基呈 非正态性分布, 图 7c表明整合素 β3亚基呈正态性分布。
图 8: 健康人群对照组中整合素 βΐ亚基的表达情况。 图 8a显示了 对照组中整合素 βΐ亚基的表达均值和基线区间,其中均值为 8.18±2.69(%), 基线区间为 2.8-13.56(%)。图 8b显示了对照组中整合素 βΐ亚基表达水平的 分布。
图 9: 健康人群对照组中整合素 β2亚基的表达情况。 图 9a显示了 对照组中整合素 β2 亚基的表达均值和基线区间, 其中表达中位数为 88.6 {%), 基线区间为 71.1-95.8 (%)。 图%显示了对照组中整合素 β2亚基表达 水平的分布。
图 10: 健康人群对照组中整合素 β3亚基的表达情况。 图 10a显示了 对照组中整合素 β3亚基的表达均值和基线区间,其中均值为 9.65±2.53(%), 基线区间为 4.59-14.71 (%)ο 图 10b显示了对照组中整合素 β3亚基表达水 平的分布。 发明详述
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在本发明中, 经研究发现含有某些 β亚基的整合素在 VTE血栓形成中 发挥着重要的作用, 是静脉血栓蛋白网络的核心蛋白。 其中所述整合素包 括 β1-3亚族的整合素。 在这一发现的基础上, 本发明提供了一种新的诊断 或者治疗静脉血栓栓塞的方法, 其中包括检测或者调节对象血液样品中的 整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平。 另外, 本发 明还涉及能够特异性地结合、 检测、 或者调节整合素 β亚基的物质, 以及 所述物质在制备用于诊断 VTE的试剂盒中的用途。
术语"血栓"是指在血管中形成的血块, 其会妨碍或阻断血流。 当血管 受损时, 血液中的血小板和某些蛋白 (如纤维蛋白、 纤维蛋白原、 血清蛋白 和细胞骨架蛋白等)会聚集形成血块来修补损伤部位。 但如果血块脱落其就 可能成为血栓。
术语"静脉血栓栓塞"是指在静脉中形成的血栓的总称, 其包括肺动脉 血栓栓塞症 (ΡΕ)和深静脉血栓形成 (DVT), 其中 ΡΕ包括急性肺栓塞 (APE)、 慢性血栓栓塞性肺动脉高压 (CTEPH)。深静脉血栓形成包括急性和慢性深静 脉血栓形成。 术语"整合素 "是指一类细胞表面受体蛋白。 此类蛋白为异二聚体, 其 含有两个不同的链, 分别称为 α亚基和 β亚基。 其中含有同一种 β亚基的 整合素构成了整合素 β亚族。 例如含有整合素 βΐ亚基 (又称作 CD29)的 βΐ 亚族、含有整合素 β2亚基 (又称作 CD18)的 β2亚族、和含有整合素 β3亚基 (又称作 CD61)的 β3亚族。其中所述 α亚基和 β亚基涵盖了由于差别剪接所 形成的变体, 例如 ITGB1的 4种变体。
本文所述"差别剪接"是指在同一个 mRNA前体中, 通过不同剪接方式 (即通过不同剪接位点组合外显子)而产生不同的 mRNA剪接异构体的过程。 通过可变剪接所获得的蛋白质产物互为同种型, 其可能会表现出不同功能 和结构特性, 或者在相同细胞中由于表达水平不同而导致不同表型。
术语"对象"是指哺乳动物个体, 其中优选为人类。
一方面, 本发明提供了一种新的诊断静脉血栓栓塞的方法。
在一个实施方案中, 本发明的方法包括检测对象血液样品中的整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平, 并将该水平的检测 结果与健康对象的对照血液样品中所测量的相应水平进行对比, 其中所述 水平的升高是用于诊断 VTE的表征。
对血液样品中的蛋白进行定量检测的方法是本领域内的常规技术。 本 领域技术人员可以根据具体需要而进行选择。 例如可以使用流式细胞术、 ELISA等基于抗体的相关方法。
术语"血液样品"是指取自对象循环***的血液组织样品,优选是取自外 周静脉的血液样品。 本发明的血液样品中可以含有血细胞和细胞外液 (即血 浆)。
本发明中的"水平升高"可以例如通过如下方法进行确定,即以一定数量 的健康人群作为对照组, 确定健康人群中的正常水平的范围, 然后如果待 测量对象的水平高于所述正常水平的范围, 则认为该待测对象的水平升高。
对于健康人群中的正常水平范围, 可以通过统计学方法来进行确定。 例如采用 Kolmogorov-Smirnov检验来进行正态性检验, P>0.05时为正 态分布。对于正态分布的数据, 以健康人群中所测量的平均值结合其标 准差 ±S)来确定正常水平范围。 对于非正态分布数据, 则以中位数结 合统计学中的 P2.5-P97.5来表示正常水平范围。
在本发明中, "水平升高"可以例如是指是指所测量的水平与健康对象血 液样品中的对照水平相比提高了 5%、 10%、 15%、 20%、 25%、 30%、 35%、 40%、 45%、 50%、 55%、 60%、 65%、 70%、 75%、 80%、 85%、 90%、 95%、 100%甚至更高。
"健康对象"是指体检正常的对象群体。 "对照水平"是指从健康对象群体 中所检测的整合素相关蛋白的水平, 然后取统计平均值并结合标准差用作 对照水平。 用于取样作为对照的健康对象群体可以例如是 50个对象、 100 个对象、 150个对象、 200个对象、 250个对象、 300个对象、 500个对象甚 至更多。
本发明的方法的步骤包括, 从待测对象中提取血液样品, 例如外周静 脉血样品。 然后利用特异性结合各种整合素 β亚基的物质例如抗体来定量 地检测所述血液样品中各种整合素 β亚基的量, 其中进行定量检测的方法 可以例如是流式细胞术、 ELISA等常规技术。提取若干 (例如 200个或更多) 健康对象的外周血样品, 用上述同样的方法来分别检测其中各种整合素 β 亚基的量, 然后取统计平均值并结合标准差用作对照水平。 将待测对象中 所测量的各种整合素 β亚基的量与相应的对照水平进行比较, 其中各种整 合素 β亚基水平的升高是用于诊断 VTE的表征。
在一个实施方案中, 本发明的方法包括检测对象血液样品中的整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的水平。 在一 个实施方案中, 本发明的方法包括检测对象血液样品中的整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的水平。 在一个实施方 案中, 本发明的方法包括检测对象血液样品中的整合素 βΐ亚基、整合素 β2 亚基、 和整合素 β3亚基的水平。
本发明中所述的静脉血栓栓塞可以是急性肺栓塞 (ΑΡΕ)、慢性血栓栓塞 性肺动脉高压 (CTEPH)、 或深静脉血栓形成 (DVT) , 优选是急性肺栓塞或者 或深静脉血栓形成。
在一个实施方案中, 本发明的方法通过使用流式细胞术来检测整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平。
"流式细胞术 "是本领域内常规的技术, 其具体操作可参见相关的文献、 教科书或者厂商的操作指弓 I。 例如可参见(Loken MR (1990). Immunofluorescence Techniques in Flow Cytometry and Sorting (2nd ed.). Wiley, pp. 341—53)。 另一方面, 本发明提供了用于特异性地检测整合素 βΐ亚基、整合素 β2 亚基、 和 /或整合素 β3亚基的水平的物质。在一个实施方案中, 本发明中提 供了特异性地结合整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的 物质, 所述物质例如是抗体、 配体和 /或拮抗剂。 在一个实施方案中, 本发 明的抗体、 配体和 /或拮抗剂是经标记的。 在一个实施方案中, 本发明的抗 体、 配体和 /或拮抗剂是经荧光标记的。在一个实施方案中, 本发明的抗体、 配体和 /或拮抗剂是经藻红蛋白 (ΡΕ)标记的。
整合素 βΐ亚基的配体例如有层粘蛋白 (LM)、 胶原蛋白 (COL)、 纤连蛋 白 (FN)、血管细胞粘附分子 -l(VCAM-l)等。整合素 β2亚基的配体例如有纤 维蛋白原 (FB)、 细胞间粘附分子 -1、 细胞间粘附分子 -2、 细胞间粘附分子 -2(ICAM-1、 ICAM-2、 ICAM-3)、 X因子、 ic3b等。 整合素 β3亚基的配体 例如有 FB、 FN、 玻连蛋白 (VN)、 von Willebrand 因子 (vWF)、 凝血酶敏感 蛋白 (TSP)等。
另一方面, 本发明涉及特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的物质在制备用于检测对象血液样品中的整合素 βΐ亚 基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平从而诊断静脉血栓栓塞的 诊断剂中的用途。
在一个实施方案中, 本发明涉及分别特异性地结合整合素 βΐ亚基、 整 合素 β2亚基、 和 /或整合素 β3亚基中至少两种的物质在制备用于诊断静脉 血栓栓塞的诊断剂中的用途。
在一个实施方案中, 本发明涉及至少两种分别特异性地结合 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的物质在制备用于诊断 静脉血栓栓塞的组合物中的用途。
在一个实施方案中, 分别特异性地结合 βΐ亚基、 整合素 β2亚基、 和 / 或整合素 β3亚基中的至少两种的物质是指至少两种不同的物质, 其中一种 物质特异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的一种, 而另一种物质特异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中 的另一种。
在一个实施方案中, 本发明涉及分别特异性地结合 βΐ亚基、整合素 β2 亚基、 和整合素 β3亚基的物质在制备用于诊断静脉血栓栓塞的组合物中的 用途。 在一个实施方案中, 分别特异性地结合 βΐ亚基、 整合素 β2亚基、 和 整合素 β3亚基的物质是指至少三种不同的物质, 其中第一种物质特异性地 结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的一种, 第二种物质特 异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的第二种, 而第 三种物质特异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的第 三种。
在一个实施方案中, 能够特异性地结合整合素 βΐ亚基、 整合素 β2亚 基、 和 /或整合素 β3亚基从而可用于特异性的检测血液样品中整合素 βΐ亚 基、 整合素 β2亚基、 和 /或整合素 β3 亚基水平的物质是抗体、 拮抗剂和 / 或配体, 优选经标记的抗体、 拮抗剂和 /或配体。 在一个实施方案中, 本发 明的抗体、 拮抗剂和 /或配体经荧光标记, 例如经藻红蛋白 (ΡΕ)标记。
另一方面, 本发明提供了用于诊断静脉血栓栓塞的试剂盒, 其含有用 于特异性地检测整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚基的水 平的物质, 其中所述物质是能够特异性地结合整合素 βΐ 亚基、 整合素 β2 亚基、 和 /或整合素 β3亚基的物质。
在一个实施方案中,本发明的试剂盒包括特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少两种的物质。
在一个实施方案中, 本发明的试剂盒包括分别特异性地结合整合素 βΐ 亚基、 整合素 β2亚基、 或整合素 β3亚基中的至少两种的物质。
在一个实施方案中, 本发明的试剂盒包括分别特异性地结合整合素 βΐ 亚基、 整合素 β2亚基、 和整合素 β3亚基的物质。
在另一个发明, 本发明还涉及整合素 βΐ亚基、 整合素 β2亚基、 和 /或 整合素 β3亚基的配体、 抗体、 拮抗剂和 /或活性抑制剂。
本发明中所用的术语 "配体"、 "抗体"、 "拮抗剂"、 "活性抑制剂"等是本 领域所常用的术语, 其含义符合本领域技术人员一般的理解, 也可以参考 常用的教科书和手册对其的定义。
在本发明中, 分别特异性地结合 βΐ亚基、 整合素 β2亚基、 和 /或整合 素 β3亚基中的至少两种的物质是指至少两种不同的物质, 其中一种物质特 异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的一种, 而另一 种物质特异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的另一 种。 而分别特异性地结合 βΐ亚基、 整合素 β2亚基、 和整合素 β3亚基的物 质是指至少三种不同的物质, 其中第一种物质特异性地结合 βΐ亚基、 整合 素 β2亚基、或整合素 β3亚基中的一种,第二种物质特异性地结合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的第二种, 而第三种物质特异性地结 合 βΐ亚基、 整合素 β2亚基、 或整合素 β3亚基中的第三种。
在另一个方面, 本发明还涉及使用本发明的方法进行诊断的装置。 例 如可以进行临床快速诊断的装置。 实施例
本发明将通过如下实施例来进行具体的描述。 这些实施例仅仅是用于 示例性的目的, 而并非对本发明的范围进行限定。 本发明具体包括如下实 施例:
实施例 1 : 用导管取样急性 VTE 血栓:
采用股静脉穿刺法, 用 7F右冠状动脉导管 (Metronic,USA)分别从一例 39岁男性急性 PE(APE)患者肺动脉内和从一例男性 50岁急性下肢股静脉内 (ADVT)取血栓长度 5-15mm、 10g的红色血拴 3-5条, 将所取得的新鲜血栓 用于下述实施例中的串联蛋白质谱分析。 实施例 2: 串联蛋白质谱检测及数据信息分析方法:
1. 方法学:
串联蛋白质谱 (MS/MS)检测及数据信息分析方法的流程图:
急性 PE、 DVT 血栓 MS/MS (型号 LTQ, Thermo Finnigan
USA)——数据库——检索蛋白——相应基因—— GO(Gene Oncology)分 析 差异基因 差异蛋白 KEGG Pathway分析一 Gene Network 分析一鉴定网络核心蛋白。
1.1 对血栓蛋白鸟枪 (shotgun)实验, 将得出的肽段数据进行生物信息 学分析, 精确鉴定肽片段所对应的蛋白, 并对该蛋白进行功能分类及生物 学通路分析.
1.2 GO分析:
GO数据库包含了基因参与的生物过程、其所处的细胞位置、其所发挥 的分子功能三方面的功能信息。 差异表达蛋白被用作 GO分析的输入。 差 异基因分别向 GO数据库的各节点映射。 计算每个节点的基因数目。 1.3 KEGG Pathway分析方法:
对差异表达蛋白进行 KEGG pathway分析。
1.4.Gene Network分析方法:
整合 3种不同的相互作用关系: (1) KEGG数据库中基因之间的蛋白互 作用、 基因调控、 蛋白修饰等关系;(2) 已有的高通量实验;(3) 已有的文献 报道中提到的基因之间的相互作用。
通 过 ROitt :〃 www.r-proiect.org/) 下 的 KEGGSOAP 软 件 包 (http://www.bioconductor.org/packages/2 ^ioc/html/KEGGSOAP.html) , 对 KEGG数据库中信号通路数据进行分析, 以鉴别基因组范围内的基因之间 的相互作用, 包括 3种关系: 酶-酶关系, 表明催化相继的反应步骤(successive reaction
ECrel
steps)的两种酶
PPrel 蛋白-蛋白相互作用, 如结合以及修饰
GErel 基因表达相互作用, 表明转录因子和靶基因产物之间的关系 蛋 白 -蛋 白 之 间 的 相 互作 用 数据可得 自 MIPS 数据 (htt :〃 mips.helmholtz-muenchen.de/proj/ppi/)。 米用共弓 I用 (co-citation)算法, 具体方法为: 下载 PubMed数据库的文献摘要, 将每个摘要分解为句子, 然 后分析每个句子中共同存在的基因名称, 即共引用的基因。 对于每对共引 用的基因对, 统计该基因对出现的频率。 基因对出现的频率越高, 则该对 基因之间存在相互作用的可能性越大。 将 PubMed数据库文献总数记作 N, 基因对中的两个基因在 PubMed文献数据库中分别独立出现的频率,分别记 作^ n。 实际基因对同时出现的次数为 fc, 基于超几何分布hypergeometric distribution), 计算出在完全随机的条件下出现大于 k次共引用的概率:
Jt-l
P = 'i-'∑lp f \ n.m,If)
a
Figure imgf000012_0001
最后基于数据结果, 将其整合为基因间的相互关系网络 (network^ 该关 系网络通过 medusa软件进行图形展示。 2. 质谱步骤及数据分析:
将实施例 1中所取得的 APE、 ADVT血栓样品分别进行质谱分析, 质谱 型号为 LTQ(Thermo Finnigan USA)。
2.1 LC-MS/MS
应用 Ettan™ MDLC*** (GE Healthcare)进行胰蛋白酶肽混合物的脱盐 以及分离。在此***中,将样品在 RP捕获柱RP trap column, Zorbax 300 SB C18, Agilent Technologies)上脱盐, 继而在 RP柱 (150 μηι i.d., 100 mm长, ColumnTechnology Inc., Fremont, CA)上分离。 选择了移动相 A(HPLC-级的 水中的 0.1%甲酸)和移动相 B (乙腈中的 0.1%甲酸)。 将 20μ§的胰蛋白酶肽 混合物上样至柱上, 通过使用 4-50% Β的线性梯度以 2μΙ7ηώι的流速进行 120分钟来完成分离。将装备有电喷射界面的 Finnigan™ LTQ™线性离子捕 获 MS (Thermo Electron)连接至所述的 LC装置用于检测洗脱的肽。 同时获 得了数据依赖性的 MS/MS波谱。每个扫描循环由如下所组成: 一个完整的 Profile模式 MS扫描、随后是 5个 Centroid模式的 MS/MS扫描,其中 Dynamic Exclusion™设置如下: 重复计数 2, 重复时间 30 s, 排除时间 90 s。 每种样 品一式三份进行分析。
2.1数据分析
使用 BioworksBrowser rev. 3.1 (Thermo Electron, San Jose, CA.)针对非冗 余 International Protein Index (IPI)人类蛋白数据库 (版本 3.26, 67687条目)对 MS/MS波谱进行自动搜寻。用 BuildSummary从 SEQUEST输出文件中提取蛋 白鉴定的结果 (Dai J, Shieh CH, Sheng QH, Zhou H, Zeng R: Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps. Anal Chem)。
将肽限制在胰蛋白酶肽的形式并且允许最多两个丢失的裂缝。 半胱氨酸 的氨甲酰甲基化被视为固定修饰, 而甲硫氨酸残基的氧化被看作是可变修 饰。 所允许的质量公差 (mass tolerance)分别是: 前体离子为 2.0 Da、 而片段 离子为 0.8 Da。蛋白的鉴定标准是基于 Delta CN (>0.1)和交叉相关得分 (Xcor 一个电荷≥1.9, 两个电荷≥ 2.2, 三个电荷≥ 3.75)。
3. 结果及分析:
将结果送交上海敏芯信息科技有限公司 (中国上海)进行如下生物信息 学分析: (来自实施例 1 中 APE患者的样品的制备、 蛋白样品鸟枪法预 分离结果见图 1, 酶解肽段、 碎片序列信息见图 2)基于数据库一检索 蛋白——鉴别相应基因——进行 Gene Oncology(GO)分析——鉴别差异基 因一鉴别差异蛋白一进行 KEGG Pathway的信号通路分析一得到蛋 白相互作用网络一鉴别网络核心蛋白。
结果:
(1) GO分析方法: 差异表达蛋白被用作 GO分析的输入。 差异基因分 别向 gene ontology数据库的各节点映射。 计算每个节点的基因数目。 统计 结果如下:
表 1 : APE血栓样品 GO分析结果 (柱状图示于图 3)
Figure imgf000014_0001
蛋白计数 富集程度 P-值 细胞循环和增殖 20 0.002718086 应激反应 40 4.62E-11 转运 52 3.74E-07
RNA代谢 14 0.999999301
DNA代谢 4 0.925590145 蛋白代谢 17 0.394000101 其它代谢过程 23 0.000119964 发育过程 6 0.183137052 细胞组构和生物发生 82 9.92E-13 细胞-细胞信号 17 0.686695527 信号转导 45 0.998147085 细胞粘附 8 2.58E-08 死亡 9 0.353211866 其它生物学过程 99 0.984977572 未知 32 0.873823656 表 2 : AVDT血栓样品 GO分析结果 (柱状图示于图 5) 功能范畴 蛋白计数 富集程度 P-值
细胞循环和增殖 6 0.091311
应激反应 28 7.98E-07
转运 32 1.90E-05
RNA代谢 4 0.999605
蛋白代谢 2 0.06044
其它代谢过程 18 0.08507
发育过程 2 0.239812
细胞组构和生物发生 43 1.21E-06
细胞-细胞信号 1 0.580049
信号转导 20 0.998072
细胞粘附 5 0.000131
死亡 9 0.46574
其它生物学过程 27 0.794559
(2) KEGG信号通路分析:将基因使用 GenMAPP v2.1向 KEGG Pathway 数据库映射, 并统计基因在每个 pathway中的富集程度 值)。对于 APE血 栓样品, 共找到 35个相关的信号通路。 而对于 AVDT血栓样品, 共找到 94个相关的信号通路。
(3) 蛋白相互作用网络分析: 同时整合 3类不同的相互作用关系来得到 蛋白相互作用的网络, 即 KEGG信号通路分析数据中的蛋白互做用、 基 因调控、 蛋白修饰等关系; (b) 已有的高通量实验, 如酵母双杂交等证实的 蛋白-蛋白相互作用; (c)已有文献报道的中提到的基因之间和蛋白之间的相 互作用。
APE血栓样品生物信息学分析结果见图 3和 4。 该结果表明 β2、 β3和 βΐ亚基整合素是红色血栓的核心蛋白。
AVDT血栓样品生物信息学分析结果见图 5和 6。 该结果表明 β3亚基 整合素和 ITGA2B (即整合素 α链 lib)是红色血栓的核心蛋白。 实施例 3 : 急性 VTE患者血栓串联蛋白质谱分析:
1. 临床测试对象的背景资料:
(1) 24例 VTE为患者组, 经影像学 (CTA,MRA,肺部通气 /灌注扫描、 血管造影或下肢静脉血管超声)检查证实患有 VTE。
其中男性 12人, 女性 12人, 年龄 25-82岁, 平均年龄 62±16岁。 8 人为急性 VTE (发病 1 个月内); 9例亚急性 VTE (发病 1-3个月内); 7 例慢性 VTE (发病 3个月以上),其中 4例为 CTEPH临床症状加重的再入 院患者。 8例急性期 VTE入院患者均采用低分子肝素皮下注射 5000单 位 /Bid,7- 10天, 停用低分子肝素的前 3-4天, 口服华法林 2.5mG/Qd; 9 例入院的亚急性期 VTE患者, 低分子肝素皮下注射 5000单位 / Qd , 7 天, 或 /和口服华法林 2.5mG/Qd; 4例再入院 CTEPH患者按亚急性期 抗凝治疗, 其他 3例慢性期的 VTE患者门诊随访, 口服华法林 2.5mG/ Qd持续治疗。 本研究检测过程中, 24例 VTE患者均未发现恶性肿瘤, 自身免疫性疾病和动脉血栓性疾病。
(2) 体检健康者 270例作为对照组。 其中男性 140人 (年龄 20-75岁),
20- 39岁组 49人, 40-59岁组 48人, 60岁以上组 43人; 女性 130人 (年龄
21- 72岁), 20-39岁组 46人, 40-59岁组 44人, 60岁以上组 40人。
PE、 DVT的诊断均有具有影像学 (CT、 MRA、 ECT或血管造影)证据和 临床表现, 标准可参照(Torbicki A, Perrier A, Konstantinides S, et al. Guidelines on the diagnosis and management of acute pulmonary embolism: the Task Force for the Diagnosis and Management of Acute Pulmonary Embolism of the European Society of Cardiology (ESC) [J]. Eur Heart J, 2008, 29(18):2276-2315; 以及 Qaseem A, Snow V, Barry P, et al. Current diagnosis of venous thromboembolism in primary care: a clinical practice guideline from the American Academy of Family Physicians and the American College of Physicians [J]. Ann Intern Med, 2007, 146(6):454-458)。
2. 整合素 β分子检测
(1)从对象中 (VTE患者、或对照)外周静脉血 2ml, 在试验管中加入 EDTA 2mg抗凝和 20μ1荧光抗体; 其中抗体分别为: 针对整合素 βΐ 亚基 (CD29) 的抗体为 ΡΕ标记,类型为鼠抗人 IgG2a,K,克隆号 HUTS-21,浓度为 50mg/L; 针对整合素 β2亚基 (CD18)的抗体为 ΡΕ标记, 类型为鼠抗人 IgGl ,K, 克隆 号 6.7, 浓度为 50mg/L; 针对整合素 β2亚基 (CD61)的抗体为 ΡΕ标记, 类 型为鼠抗人 IgGl,K, 克隆号 VI-PL2 , 浓度为 50mg/L; CD29-PE(pi) , CD18-PE(P2), CD61-PE(P3)荧光抗体可得自美国 BD公司 (Becton, Dickinson and Company, 美国);
(2)加样后充分混匀于室温下避光反应 30分钟;
(3)在试验管中加入溶血素 500μ1; 其中所使用的溶血素用于溶解红细 胞, 型号为 OptiLyse C, 可得自美国 Beckman-Coulter公司。
(4) 37°C水浴 30分钟;
(5)洗涤后加入 ΙΟΟμΙ缓冲液 (0.01M的 PBS, ΡΗ7.2), 进行流式细胞仪 检测。 其中使用美国 BECKMAN-COULTER公司 EPICS XL-4型流式细胞 仪;
3. 细胞免疫及整合素流式检测操作流程:
(1) 实验时在试管中加入 EDTA 2mg抗凝血, 同时设置同型对照管, 按 荧光标记不同分别加入 20μ1小鼠 IgGl-PC5、 IgGl-FITC, IgGl-PE(CD29分 子检测加入 20μ1小鼠 IgG2-PE, 50mg/L), 再分别加入相应的荧光抗体 20μ1 (50mg/L)。 充分混匀后至于室温下避光反应 30分钟。 然后加入溶血素型号 OptiLyse C (美国 BECKMAN-COULTER公司产品) 500μ1, 置于 37°C水浴箱 反应 30分钟。取出后经过洗涤, 每管加入 500μ1鞘液 (含活性成分为硫酸钠 9.84g/L,氯化钠 4.07 g/L,盐酸普鲁卡因 0.11 g/L),得自美国 Beckman-Coulter 公司。 随后进行流式细胞仪 (美国 BECKMAN-COULTER公司产品, 型号 EPICS XL-4)检测。
(2) 以 BECKMAN-COULTER标准荧光微球调节流式细胞仪 PMT 电 压、 荧光补偿、 灵敏度, 设定检测方案。 每管收集 10000个细胞。 以同型 对照管散点图中对应细胞群设门, 根据标记的荧光所相对应的象限确定阳 性细胞百分比 (%)。 使用流式细胞仪自带的 SYSTEM-Π软件对结果进行分 析。
4.统计学分析: 对照组采用 SPSS18统计学软件进行统计分析。 正态 性检验采用 Kolmogorov-Smirnov检验, P>0.05时为正态分布。 正态分 布数据以 ±S 表示, 不同年龄段组间比较采用方差分析, PO.05 表示 差异有统计学意义。 非正态分布数据以中位数和 P2.5-P97.5表示, 不同 性别组间比较采用 Wilcoxon秩和检验, PO.05表示差异有统计学意义; 不同年龄组间比较采用 Kmskal-Wallis H检验, PO.05表示差异有统计 学意义。
对于正态分布的数据,以健康人群中所测量的平均值结合其标准差 ±s)来确定正常水平范围。 对于非正态分布数据, 则以中位数结合统 计学中的 P2.5-P97.5来表示正常水平范围。
VTE组 β1、 β2和 β3与对照组比较, 高于正常参考区间值视为表 达增加。
5. 结果:
结果以总细胞计数中显示含有相应整合素亚基的细胞(阳性细胞) 的百分比来表示 (即, 以 10000个细胞为单位, 阳性细胞占的百分比)。 参见图 7-10以及下表 3。 270例对照组, 整合素 β1、 β3亚基呈正态性 分布, 整合素 β2亚基呈非正态性分布。 不同性别之间结果比较无显著 性差异, 随年龄增加 β1、 β3 有下降趋势; 整合素 βΐ 亚基基线区间为 2.8-13.56%, 整合素 β2亚基基线区间为 71.1-95.8%; 整合素 β3亚基基 线区间为 4.59-14.71%。
表 3: VTE患者的信息及检测结果
序号 诊断 β 1 β2 β3 D-di CRP Fibr inogen 性别 年龄 诊断 类别
方法 2.8-13.56% 70.1-95.8% 4.59-14.71% <0.3mg/L <2.87mg/L 2-4.5g/L
1 男 82 下肢 DVT 亚急性期 超声 11.37 97.5† 14.5 1.06† 132† 4.613†
2 男 56 PE/下肢 DVT 亚急性期 CTA/超声 6.81 76.3 15.9† 0.47† 20.8† 4.761†
3 男 33 PE 亚急性期 CTA 11.4 75 18.9† 0.22 51.8† 4.553†
4 女 58 下肢 DVT 慢性期 超声 21.6† 66.8 8.63 0.05 2.863
5 女 66 CTEPH 慢性期 CTA 12.8 96.4† 12.9 0.05 0.3 3.25
6 女 54 下肢 DVT 亚急性期 超声 18.5† 85 18.8† 0.7† 0.7 2.56
7 女 56 下肢 DVT 慢性期 超声 10.1 94.7 16† 0.05 0.7 3.65
8 男 68 CTEPH 慢性期 CTA 7.27 98.2† 12.3 0.14 24.7† 3.825
9 女 25 PE 亚急性期 CTA 5.8 94.2 16.9† 0.35† 36† 2.761†
10 女 82 PE 亚急性期 1 CTA 17.3† 99.5† 5.6 0.05 0.2 2.495
11 男 65 下肢 DVT 亚急性期 超声 14.5† 96.8† 9.85 0.05 1.7 3.04
12 女 61 PE 急性期 CTA 4.5 96.4† 17.3† 2.495
13 男 71 PE 急性期 PAG 14.3† 95.6 13.1 1.48† 16.4† 3.761
14 女 81 PE 慢性期 CTA 10.2 96.7† 7.4 0.08 3.383
15 男 49 PE 急性期 CTA 15.4† 90.1 14.6
16 女 53 PE 急性期 肺通气 /灌注 13.1 97.2† 6.4 1.25† 0.6 3.576
17 男 71 PE 急性期 CTA 17.52† 98.2† 19† 0.24 0.7 2.714
18 男 80 CTEPH 慢性期 通气 /灌注扫描 23.5† 99.4† 18.7† 0.75† 32† 3.264
19 女 75 PE 急性期 通气 /灌注扫描 6.27 94.7 15.4† 0.64† 2.6 2.11
20 女 78 PE 急性期 通气 /灌注扫描 6.92 96.9† 13.4 0.96† 38† 4.481
21 男 64 下肢 DVT 亚急性期 超声 14.5† 96.8† 9.85 0.05 3.04
22 女 80 CTEPH 慢性期 CTA 17.3† 99.5† 5.6 0.09 2 2.607
23 男 48 PE 急性期 CTA 15.49† 90.1 14.6 0.89† 4† 2.00
24 男 32 PE 亚急性期 CTA 11.4 75 18.9† 0.25 92† 4.533† 合计: 62±16 12.83±5.07 91.96±9.28 13.52±4.94
4例 CTEPH患者, 心脏超声收缩压 65— 74mmHg之间; 序号 4、 1、 14号为门诊随访患者
本研究 24例急性期、 亚急性期和慢性期的 VTE患者, 尽管抗凝方案 不同, 抗凝时间长短不同, 24例 VTE患者整合素 β亚基均单项或联合表达 高于健康人群区间的上限值。 其中 15/24例 (62.5%)VTE患者整合素 β亚基 单项表达升高, 11/24例 (45.8%)整合素 βΐ亚基升高, 急性期 4/11例, 亚急 性期 4/11例, 慢性期 3/11例; 13/24例 (54.2%)整合素 β2亚基升高, 急性期 4/13例, 亚急性期 4/13例, 慢性期 5/13例; 10/24例 (41.7%)整合素 β3亚基 升高, 急性期 3/10例, 亚急性期 6/10例, 慢性期 1/10例。 9/24例整合素 β 亚基两项以上联合升高 (37.5%), 其中 β1,β2和 β3共同升高 3/24例 (12.5%), βΐ和 β2共同升高 6/24例 (25%), βΐ和 β3共同升高 1/24例 (4.2%), β2和 β3 共同升高 1/24例 (4.2%)。上述细胞学检测结果表明 VTE患者整合素 β亚族 高表达可用于对 VTE进行诊断。 上述实施例中的结果表明, 健康人群血细胞整合素 β1、 β2和 β3亚基的 表达是可以检测的, βΐ和 β3亚基的表达呈正态分布, β2亚基的表达呈非 正态分布。 性别之间无统计学差异。 随年龄增加 βΐ和 β3亚基的表达呈下 降趋势。 本发明中 8例急性期、 9例亚急性期和 7例慢性期的 VTE患者, 整合素 β1、 β2和 β3亚基分别或联合表达升高, 均高于对照组的参考区间 上限,与血栓串联蛋白质谱信息分析的结果吻合。本研究 VTE组整合素 β1、 β2、 和 /或 β3 亚基的表达分别或联合显著增高, 表明整合素活化和信号转 导作用增强, VTE患者整合素 β1、 β2、 β3亚基在持续抗凝治疗过程中的高 表达, 表明其稳定性, 敏感性和特异性, 同时也表明患者仍处于易栓状态。

Claims

权 利 要 求 书
1. 特异性地结合整合素 βΐ亚基、整合素 β2亚基、和 /或整合素 β3亚 基的物质在制备用于检测对象血液样品例如外周静脉血样品中的整合素 βΐ 亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平从而诊断静脉血栓栓塞 (VTE)的诊断剂中的用途。
2. 权利要求 1 的用途, 其中所述诊断剂包括至少一种分别特异性地 结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少一种 的物质, 其中优选所述诊断剂包括分别特异性地结合整合素 βΐ亚基、 整合 素 β2亚基、 和整合素 β3亚基的物质。
3. 权利要求 1 的用途, 其中所述静脉血栓栓塞是急性肺栓塞 (ΑΡΕ)、 慢性血栓栓塞性肺动脉高压 (CTEPH)、或深静脉血栓形成 (DVT), 优选是急 性肺栓塞或急性深静脉血栓形成。
4. 权利要求 1-3中任一项的用途, 其中所述物质为抗体、 配体、 和 /或 拮抗剂, 优选是经标记的抗体、 配体、 和 /或拮抗剂。
5. 权利要求 4的用途, 其中所述抗体、 配体、 和 /或拮抗剂是经荧光标 记的, 例如经藻红蛋白 (PE)标记。
6. 一种检测对象血液样品例如外周静脉血样品中的整合素 βΐ 亚基、 整合素 β2亚基、 和 /或整合素 β3亚基的水平从而诊断静脉血栓栓塞 (VTE) 的试剂盒, 其含有分别特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 / 或整合素 β3亚基的物质。
7. 权利要求 6的试剂盒, 其中包括至少两种分别特异性地结合整合素 βΐ亚基、 整合素 β2亚基、 和 /或整合素 β3亚基中的至少一种的物质, 其中 优选所述试剂盒中包括分别特异性地结合整合素 βΐ亚基、整合素 β2亚基、 和整合素 β3亚基的物质。
8. 权利要求 6的试剂盒, 其中所述静脉血栓栓塞是急性肺栓塞 (APE)、 慢性血栓栓塞性肺动脉高压 (CTEPH)、或深静脉血栓形成 (DVT), 优选是急 性肺栓塞或急性深静脉血栓形成。
9. 权利要求 6-8中任一项的试剂盒, 其中所述物质为抗体、 配体、 和 / 或拮抗剂, 优选是经标记的抗体、 配体、 或拮抗剂。
10. 权利要求 9的试剂盒, 其中所述抗体、 配体、 和 /或拮抗剂是经荧 光标记的, 例如经藻红蛋白 (PE)标记。
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